Enzyme immunoassay with a macroporous hydrophobic synthetic polymer cloth containing an immobilized antibody or antigen

ABSTRACT

An immunoassay device containing an immobilized antibody or antigen is provided by directly absorbing and absorbing an unmodified antibody or antigen on and within a woven or non-woven macroporous hydrophobic synthetic polymer cloth formed of a synthetic polymer selected from the group consisting of plypropylene, polyester, nylon and polyethylene. The cloth has a thickness of more than about 200 μm and contains pores in the form of spaces between fibers exceeding about 20 μm in diameter, and has a Frazier Air Permeability in CFM/ft 2  at 0.5&#34; H 2  O of about 215 for a cloth of thickness of about 40 mils. The cloth has a large surface area for binding to an antibody or antigen and can accommodate a large volume of liquid per surface area and has minimum flow resistance. The cloth containing an immobilized antibody or antigen may be used to carry out an enzyme immunoassay by contacting the cloth with a sample containing an antigen or antibody, incubating the cloth with an enzyme-antibody conjugate and then reacting enzyme bound to the cloth with a chromogenic substrate-indicator to produce a visible color. Other immunoassay embodiments may also be carried out and a control cloth can be used such that a difference in color from that obtained with the control cloth determines the amount of antigen or antibody present in a sample.

BACKGROUND OF THE INVENTION

(i) Related Invention

This invention is a continuation-in-part of co-pending application Ser.No. 099,393 filed Sept. 21, 1987, now abandonedthe entire contents ofwhich are incorporated herein by reference.

(ii) Field of the Invention

This invention relates to the detection of antigens or haptens based onimmunoassay techniques.

An antigen is an extraneous substance which, when introduced into thebody of vertebrates, causes the production of an antibody which canspecifically complex with that antigen. Any substance, for example aprotein, which is not normally present in certain organisms, can causethe formation of antibodies when it infiltrates into or is applied to anorganism under suitable conditions. An antibody once produced is alsocapable of binding a hapten, i.e., a relatively small and simplecompound which may be the determinant group of a given antigen. Thehapten is capable of binding with the specific antibody but is incapableitself of giving rise to the production of an antibody, unless it isbound to an antigenic carrier. These small molecular weight antigens(haptens) may require conjugation with large molecular weight carriersin order to elicit antibody production. This antigen-antibody complexingis the basis of immunoassays.

The binding interaction between an antigen or a hapten and its antibodyis specific and sensitive. Other types of materials that participate insimilar specific and sensitive binding interactions are: enzymes andtheir substrates; hormones; vitamins; metabolites; and pharmacologicalagents; and their receptors and binding substances.

Since virtually any foreign compound can be made immunogenic, the domainof immunoassays is unlimited.

(iii) Description of the Prior Art

Diagnostic tests claim a large share of the health care market. In bothhuman and veterinary medicine, the definitive diagnosis of infectiousdiseases require the demonstration of the infectious agents or theircomponents. Traditional cultural methods for the detection of pathogensare slow, expensive and of uncertain sensitivity, and require extensivelaboratory facilities. To overcome some of these disadvantages, specificbinding assay techniques have provided analytical methods fordetermining various organic substances of diagnostic, medical,environmental and industrial importance which appear in liquid mediumsat very low concentrations. Specific binding assays are based on thespecific interaction between the ligand, i.e. the bindable analyte underdetermination, and a binding partner therefor. When one of the ligandand its binding partner is an antibody and the other is a correspondinghapten or antigen, the assay is known as an immunoassay. In additionseveral immunological tests are now commercially available, namely:agglutination tests; immunofluorescent tests; and enzyme immunoassays.However, many of these tests require the use of microscopes,spectro-photometers, or other laboratory facilities, limiting their useunder field conditions. Prompt and effective control of diseases dependson rapid and simple field tests.

Radioimmunoassay employs a radioactive isotope as the label. Such anassay necessarily must follow the heterogeneous format since themonitoraole character of the label is qualitatively unchanged in thefree- and bound-species. Because of the inconvenience and difficulty ofhandling radioactive materials and the necessity of a separation step,homogeneous assay systems have been devised using materials other thanradioisotopes as the label component, including enzymes, bacteriophages,metals and organometallic complexes, coenzymes, enzyme substrates,enzyme activators and inhibitors, cycling reactants, organic andinorganic catalysts, prosthetic groups, chemiluminescent reactants, andfluorescent molecules. Such homogeneous specific binding assay systemsprovide a detectable response, e.g., an electromagnetic radiationsignal, e.g. chemiluminescence, fluorescence emission, or color change,related to the presence of amount of the ligand under assay in theliquid sample.

Immunoassays diagnose infectious diseases by detecting either increasedtiters of antibodies against pathogen antigens or the presence of thepathogens or their antigens. Antigen assays offer more definitivediagnosis of infectious diseases as the capacity to produce antibodiesremains in subjects which have recovered from the disease or havepreviously been vaccinated.

Enzyme immunoassays use enzyme-labeled immunoreagents (antibodies orantigens) for the detection of captured antigens or antibodies capturedin a solid phase.

Adsorption onto an easily recoverable solid phase is a simple and rapidmeans of immobilization of immunoreactants for the subsequent capture ofantigens or antibodies from a test sample. Since antibodies and manyantigens contain hydrophobic regions in their structures, they bindreadily to hydrophobic surfaces. Most commonly used enzyme immunoassaysdepend on the adsorption of immunoreactants onto either a flat surfaceor membrane. Solid phases, e.g. microtiter plates, tubes or beads, andplastics, e.g. polystyrene, polyvinyl chloride, nylon, andpolymethacrylate have commonly been used. Although nitrocellulosemembranes have been used to adsorb antigens as well as antibodies, theseare thin and can only accommodate a small volume of test sample whichhas a limited contact with a surface area. Furthermore, since their poresizes are small, their effective washing requires a vacuum suctionapparatus which holds them airtight.

The patent literature is replete with descriptions of techniques andmeans for effecting immunoassays. A representative selection of suchpatents include the following:

1) Canadian Patent 1,031,257 issued May 16, 1978 to R. Dietrich for"Carrier for Immunological Determinations" (directed to a devicecomprising an immunologically-reactive material on an object carrier ora film, the immunologically-reactive material being in a lyophilised andself-adhering form);

2) Canadian Patent No. 1,060,342 issued Aug. 14, 1979 to O. Lostia et alfor "Fibres Incorporating Antibodies, Antigens and Antisera, Method ForTheir Preparation and Their Use" (directed to a polymeric structurecomprising a porous artificial fibre where the substance occluded in thefibre was antibodies, antigens or antisera, and where the pores of thefibre were of such nature as to prevent escape of the occluded substancebut to allow for the penetration of the agent that is to be reacted withthat substance);

3) Canadian Patent No. 1,083,036 issued Aug. 5, 1980 to G. Bolz for"Indirect Solid Surface Test For Antigens or Antibodies", (directed to aspecifically-described procedure for determining reacted labeledantibody);

4) Canadian Patent No. 1,107,195 issued Aug. 18, 1981 to D. Wagner et alfor "Specific Binding Assay Employing Polystyrene As Separating Agent"(which provided a specific binding assay method using nonion-exchangecross-linked polystyrene for determining a ligand in, or theligand-binding capacity of, a liquid medium);

5) Canadian Patent No. 1,108,986 issued Sept. 15, 1981 to D. Wagner etal for "Specific Binding Assay Employing Polyvinyl Alcohol As SeparatingAgent" (which provided a specific binding assay method usingnonion-exchange cross-linked polyvinyl alcohol for determining a ligandin or the ligand binding capacity of a liquid medium);

6) Canadian Patent No. 1,152,430 issued Aug. 23, 1985 to J. Gordon et alfor "Protein On Nitrocellulose Sheet Support" (directed to a solidsupport for proteins consisting of a porous nitrocellulose sheetcontaining an electrophoretically transferred replica of anelectropherogram of proteins in a gel);

7) Canadian Patent No. 1,199,269 issued Jan. 14, 1986 to V. A.Marinkovitch for "Multiple Component Binding Assay System and Method ofMaking and Using it" (directed to a diagnostic kit which included asupport having a plurality of cotton threads supported in apredetermined spaced relation for simultaneous contact with a liquidtest sample);

8) U.S. Pat. No. 3,951,741 patented Apr. 20, 1976 by R. F. Devlin for"Sensitized Matrix For Detection of Disease" (directed to a specificsensitized matrix for diagnosing both infectious and non-infectiousdiseases, including an insoluble, inert, pliable and wettable matrixhaving a network of pores, and a protein polymer network immobilized inthat network of pores);

9) U.S. Pat. No. 4,168,146 patented Sept. 18, 1979 by A. O. Grubb et alfor "Immunoassay With Test Strip Having Antibodies Bound Thereto"(directed to a diagnostic test device useful for immunochemicalquantification, which was a carrier strip comprising a silica-modifiedmicro-porous polymer having finely-divided silicasubstantially-uniformly embedded in a particularly-recited permeable,continuous polymeric matrix);

10) U.S. Pat. No. 4,277,561 patented Jul. 7, 1981 by D. Monget et al for"Support For The Determination of Enzyme Activities and Process"(directed to a support for the determination of enzyme activity in abiological extract wherein the support comprised a fibrous materialimpregnated with a substrate and a particularly-recited water-soluble pHstabilizer);

11) U.S. Pat. No. 4,347,311 patented Aug. 31, 1982 by H. H. Schmitz for"Enzyme Immunoassay For Determining Antigen Specific Antibodies and TestKit For Carrying Out This Assay" (directed to a highly sensitive enzymeimmunoassay procedure for determining antibodies which are specific toantigens by coating a particularly-recited solid support with anantibody); and 12) U.S. Pat. No. 4,442,204 patented Apr. 10, 1984 by A.C. Greenquist for "Homogeneous Specific Binding Assay Device andPreformed Complex Method" (directed to a test device comprising a solidcarrier member, e.g., a fibrous web matrix, e.g. paper, or a polymericfilm or gel, incorporated with specifically-recited reagents for ahomogeneous specific binding assay system).

SUMMARY OF THE INVENTION Aims of the Invention

Accordingly, those concerned with the development and use of immunoassaytechniques and related devices have recognized the desirability forfurther improvements and it is therefore one object of the invention toprovide a rapid, accurate method for the quantitation of antigen orantibody on a solid surface.

A further object of the invention is to provide a method to providerapid and sensitive immunoassays.

A still further object of the present invention is the provision of arelatively simple yet high effective and sensitive diagnostic test forthe detection of specific disease states, both infectious andnon-infectious.

Yet another object of this invention is the use of the macroporoushydrophobic cloths to make enzyme immunoassays rapid and simple.

Yet another object of this invention is to enable the use of macroporoushydrophobic cloths for enzyme immunoassays for antigens and haptens aswell as for antibodies.

STATEMENTS OF INVENTION

By this invention, an enzyme immunoassay device is provided comprisingthe combination of (a) a macroporous hydrophobic synthetic polymer wovenor non-woven cloth having a thickness of more than about 200 μm andhaving spaces between fibres exceeding about 20 μm in diameter, thecloth consisting entirely of unmodified hydrophobic threads formed of asynthetic polymer selected from the group consisting of polypropylene,polyester, nylon, and polyethylene; and (b) an unmodified antibody or anunmodified antigen directly adsorbed thereon and directly absorbedtherein; the cloth having a Frazier Air Permeability in CFM/ft² at0.5"H₂ O of about 215 for a cloth of thickness about 40 mils, the cloththereby having such porosity that it can accommodate a large volume ofliquid per surface area thereof, that it has a large surface area forbinding to the antibody or the antigen, respectively, and that it hasminimum flow resistance.

The term "macroporous" as applied to cloths when used herein is intendedto mean textiles composed of hydrophobic synthetic polymeric fibers,which are either woven or non-woven into a physically structurallystable cloth of more than about 200 μm thickness, such that the pores(i.e., spaces between the fibers) exceed about 20 μm in diameter.

The term "hydrophobic" as applied to cloths when used herein is intendedto mean that the cloths repel water, the degree of repelling beingdependent on the pore size and the inherent polymeric properties.

The term "unmodified" when referring to the hydrophobic threads isintended to mean that the threads have not been subjected to anychemical treating reaction, nor to any surface coating treatment.

The term "unmodified" when referring to the antibody or the antigens isintended to mean that neither the antibody nor the antigen have beensubjected to any chemical treating reaction for the purpose, e.g. ofenabling them to adsorb to the surface of the macroporous hydrophobiccloth.

The term "non-woven" when referring to the cloth is intended to mean acloth formed from a random arrangement of natural or synthetic fibres byadhesives, heat and pressure, or needling techniques.

This invention also provides an enzyme immunoassay method for detectingan antigen comprising the steps of: a) treating a surface of animmunoassay device comprising a macroporous hydrophobic syntheticpolymer woven or non-woven cloth having a thickness of more than about200 μm and having spaces between fibres exceeding about 20 μm indiameter, the cloth consisting entirely of unmodified hydrophobicthreads formed of a synthetic polymer selected from the group consistingof polypropylene, polyester, nylon, and polyethylene with an unmodifiedantibody, thereby to have an antibody directly adsorbed thereon anddirectly absorbed therein, the cloth having a Frazier Air Permeabilityin CFM/ft² at 0.5"H₂ O of about 215 for a cloth of thickness about 40mils, the cloth thereby having such porosity that it can accommodate alarge volume of liquid per surface area thereof, that it has a largesurface area for binding to the antibody and that it has minimum flowresistance, thereby to provide an antibody surface-treated cloth; b)applying, to the surface of the antibody surface-treated cloth, anantigen being assayed, thereby to provide an antigen-treated cloth; c)incubating the antigen-treated cloth with a sample to be tested for theantigen, thereby to provide an incubated cloth; d) washing the incubatedcloth with a buffer to remove unadsorbed material, thereby to provide awashed cloth; e) incubating the washed cloth with an enzyme-antibodyconjugate prepared by coupling purified antibody specific for theantigen to a suitable indicator enzyme, thereby to provide an incubatedwashed cloth; f) washing the incubated washed cloth with a buffer toremove unreacted conjugate; and g) detecting enzyme-antibody conjugateremaining thereon by incubation in a chromogenic substrate indicatorsolution to produce a visible colour upon product formation, indicativeof the presence of an antigen.

This invention also provides an enzyme immunoassay method for detectingan antibody comprising the steps of: a) treating a surface of animmunoassay device comprising a macroporous hydrophobic syntheticpolymer woven or non-woven cloth having a thickness of more than about200 μm and having spaces between fibres exceeding about 20 μm indiameter, the cloth consisting entirely of unmodified hydrophobicthreads formed of a synthetic polymer selected from the group consistingof polypropylene, polyester, nylon, and polyethylene with an unmodifiedantigen, thereby to have an antigen directly adsorbed thereon anddirectly absorbed therein, the cloth having a Frazier Air Permeabilityin CFM/ft² at 0.5"H₂ O of about 215 for a cloth of thickness about 40mils, the cloth thereby having such porosity that it can accommodate alarge volume of liquid per surface area thereof, that it has a largesurface area for binding to the antigen and that it has minimum flowresistance; thereby to provide an antigen surface-treated cloth; b)applying, to the surface of the antigen surface-treated cloth, anantibody being assayed, thereby to provide an antibody-treated cloth; c)incubating the antibody-treated cloth with a sample to be tested for theantibody, thereby to provide an incubated cloth; d) washing theincubated cloth with a buffer to remove unadsorbed material, thereby toprovide a washed cloth; e) incubating the washed cloth with anenzyme-antibody conjugate prepared by coupling purified antibodyspecific for the antibody to a suitable indicator enzyme, thereby toprovide an incubated washed cloth; f) washing the incubated washed clothwith a buffer to remove unreacted conjugate; and g) detectingenzyme-antibody conjugate remaining thereon by incubation in achromogenic substrate indicator solution to produce a visible colourupon product formation, indicative of the presence of an antibody.

This invention also provides an enzyme immunoassay method for detectingan antibody comprising the steps of: a) treating an immunoassay devicecomprising a macroporous hydrophobic synthetic polymer woven ornon-woven cloth having a thickness of more than about 200 μm and havingspaced between fibres exceeding about 20 μm in diameter, the clothconsisting entirely of unmodified hydrophobic threads formed of asynthetic polymer selected from the group consisting of polypropylene,polyester, nylon, and polyethylene with an unmodified antibody, therebyto have an antigen directly adsorbed thereon and directly absorbedtherein, the cloth having a Frazier Air Permeability in CFM/ft² at0.5"H₂ O of about 215 for a cloth of thickness about 40 mils, the cloththereby having such porosity that it can accommodate a large volume ofliquid per surface area thereof, that it has a large surface area forbinding to the antibody and that it has minimum flow resistance, therebyto provide an antibody surface-treated cloth; b) applying to the surfaceof the antibody surface-treated cloth, a mixture of the antibody beingassayed and an enzyme-antigen conjugate specific for the antibodyadsorbed onto the cloth, thereby to provide an antibody/enzyme-antigenconjugate treated cloth; c) applying, to the surface of a control clothidentical to the hydrophobic synthetic polymer cloth, the sameenzyme-antibody conjugate, thereby to provide an enzyme-antigenconjugate treated control cloth; d) incubating both theantibody/enzyme-antibody conjugate treated cloth and the enzyme-antigenconjugate treated control cloth substantially simultaneously, to provideincubated cloths; e) washing both the incubated antibody/enzyme-antigenconjugate treated cloth and the incubated enzyme-antigen conjugatetreated control cloth with a buffer solution; and f) detecting theantibody by incubation of both the washed antibody/enzyme-antigenconjugate treated cloth and the washed enzyme-antigen conjugate treatedcontrol cloth in a chromogenic substrate indicator solution to produce avisible colour upon product formation indicative of the presence ofantibody, the amount of antibody being determined by the difference inintensity of the colour on the antibody/enzyme-antigen conjugate treatedcloth and the colour on the enzyme-antigen conjugate treated controlcloth.

This invention also provides an enzyme immunoassay method for detectingan antigen comprising the steps of: a) treating an immunoassay devicecomprising a macroporous hydrophobic synthetic polymer woven ornon-woven cloth having a thickness of more than about 200 μm and havingspaced between fibres exceeding about 20 μm in diameter, the clothconsisting entirely of unmodified hydrophobic threads formed of asynthetic polymer selected from the group consisting of polypropylene,polyester, nylon, and polyethylene with an unmodified antibody, therebyto have an antibody directly adsorbed thereon and directly absorbedtherein, the cloth having a Frazier Air Permeability in CFM/ft² at0.5"H₂ O of about 215 for a cloth of thickness about 40 mils, the cloththereby having such porosity that it can accommodate a large volume ofliquid per surface area thereof, that it has a large surface area forbinding to the antibody and that it has minimum flow resistance, therebyto provide an antibody surface-treated cloth; b) applying to the surfaceof the antibody surface-treated cloth a mixture of the antigen beingassayed and an enzyme-antibody conjugate specific for the antigen,thereby to provide an enzyme-antibody conjugate treated cloth; c)applying, to the surface of a control cloth identical to the hydrophobicsynthetic polymer cloth, the same enzyme-antibody conjugate, thereby toprovide an enzyme-antibody conjugate treated control cloth; d)incubating both the antigen/enzyme-antibody conjugate treated cloth andthe enzyme-antibody conjugate treated control cloth substantiallysimultaneously to provide incubated cloths; e) washing both theincubated antigen/enzyme-antibody conjugate treated cloth and theincubated enzyme-antibody conjugate treated control cloth with a buffersolution; and f) detecting the antigen by incubation of both theantigen/enzyme-antibody conjugate treated cloth and the enzyme-antibodyconjugate treated control cloth in a chromogenic substrate indicatorsolution to produce a visible colour upon product formation, indicativeof the presence of antigen, the amount of antigen being determined bythe difference in intensity of the colour on the antigen/enzyme-antibodyconjugate treated cloth and the colour on the enzyme-antibody conjugatetreated control cloth.

OTHER FEATURES OF THE INVENTION

By another feature of this invention, the enzyme immunoassay device maybe so-treated with an antiserum containing an antibody specific for theantigen being tested, or by still another feature of this invention, itmay be so-treated with a purified antibody bearing the appropriatespecificity. By yet another feature of this invention, the antibodiespresent in the antiserum may be partially denatured prior to beingso-applied to the hydrophobic cloth, e.g. by exposure to a low pHenvironment, e.g, a pH of about 2.5, or by heating. Alternatively, byyet a further feature of this invention, the antibodies may beaffinity-purified prior to being so-applied to the hydrophobic cloth. Bystill another feature of this invention, the antibody may be provided bydiluted antiserum.

By yet another feature of this invention, the enzyme immunoassay deviceof the invention may be in the form of the macroporous hydrophobic clothbonded to a different material, thereby to provide an antibody-coated orantigen-coated test strip that may be handled throughout an assayprocedure. Thus, the present invention embraces the bonding, in anysuitable manner, of the so-treated macroporous hydrophobic cloth to adipstick.

GENERALIZED DESCRIPTION OF THE INVENTION

It has surprisingly been found that all macroporous hydrophobic cloths(as defined above) when coated with an unmodified antigen or antibody(as defined above) provide an unexpected improvement in the enzymeimmunoassay procedure of the present invention. All such macroporoushydrophobic cloths, whether they be formed of polypropylene, nylon,polyester or polyethylene threads, have the following characteristics:they can accommodate a larger volume of sample per area; they have alarger surface area for binding immunoreactants and for immunoreactions;they are easily washed because of minimum flow resistance; and they haveboth strength and durability. Thus, it has been found that themacroporous hydrophobic cloths used as supports in the enzymeimmunoassay methods of this invention provide a large volume forabsorption per surface area for antigen-antibody interaction.Macroporous polypropylene, polyethylene, nylon, and polyester cloths, byvirtue of their hydrophobic characteristics, have been found to adsorband absorb antibodies and thus provide a large surface area for antigencapture. Macroporous cloths all have such minimum flow resistance.

Such macroporous hydrophobic cloths made of, e.g. polypropylene andpolyester, are readily commercially available and are moderately pricedbecause of their large commercial demand as textiles and filters.Macroporous 100% nylon cloth is commercially available as a genericproduct and was acquired locally in the Ottawa, Canada area. Macroporouswoven polyester cloth is commercially available as a generic product andwas acquired locally in the Ottawa, Canada area. Macroporous non-wovenpolypropylene filter cloth is available as a generic product and waspurchased from Aldrich Chemical Co. A variety of non-woven, macroporouspolyester cloths were obtained from DuPont, and is known by thetrademark SONTARA.

One preferred embodiment of such SONTARA is SONTARA 8100™, which has thefollowing chemical and physical characteristics.

Typical Physical Properties of SONTARA 8100 are:

    __________________________________________________________________________                           SHEET GRAB                                                                            TRAPEZOID     FRAZIER AIR                                                                             ROLL SIZE                      UNIT           TENSILE TEAR    MULLEN                                                                              PERMEABILITY                                                                            (7" ID CORE)                   WEIGHT THICKNESS                                                                             (lbs)   (lbs)   BURST (CFM/ft.sup.2                                                                           in. lin.                       (oz/yd..sup.2)                                                                       (mils)  MD  XD  MD  XD  (psi) @ 0.5" H.sub.2 O)                                                                       O.D.                                                                              yds.               __________________________________________________________________________    Style   40     40      70  45  35  40  120   215       44  1700               100% Polyester                                                                8100                                                                          __________________________________________________________________________

Frazier Air Permeability is described in ASTM D737 75, the contents ofwhich are hereby incorporated by reference.

It has been found that the use of macroporous hydrophobic cloths alsoprovide improvements in the sensitivity of antiserum-based enzymeimmunoassay. These improvements are consequence of an increase in thehydrophobicity of the denatured Fc region of the antibodies, which inturn causes these to be adsorbed more strongly to the hydrophobic clothsurface and in greater numbers. The partially-denatured state may alsoensure that the antibodies adhere to the solid phase in a more idealorientation, with the Fc region.

Consequently, the enzyme immunoassay procedure of one embodiment of thisinvention may consist of the following: a macroporous hydrophobic clothhaving such porosity that it can absorb and adsorb a large volume ofliquid per surface area of the cloth, is treated to have adsorbed andabsorbed thereon and therein, either an antiserum containing antibodyspecific for the antigen being tested, or a purified antibody bearingthe appropriate specificity, and is subsequently incubated with the testsample purported to contain the antigen. The macroporous hydrophobiccloth is then washed with an appropriate buffer to remove any unadsorbedand unabsorbed material, and is then incubated with an enzyme-antibodyconjugate prepared by coupling purified antibody specific for theantigen to a suitable indicator enzyme. The macroporous hydrophobiccloth is then washed with buffer to remove unreacted conjugate, and theremaining conjugate is detected by incubation in a chromogenicsubstrate-indicator solution which produces a visible colour uponproduct formation.

The enzyme immunoassay procedure of another embodiment of the inventionmay consist of the following: a test sample containing the antigen to beassayed is mixed with a suitable enzyme-antibody conjugate, e.g. dilutedhorseradish peroxidase (HRP)-antibody conjugate specific for the antigenof interest, and an aliquot of this mixture is incubated with anantigen-treated hydrophobic cloth. A control macroporous hydrophobiccloth is treated with a mixture of the same enzyme-antibody conjugatebut without the free antigen.

After washing with a suitable buffer solution, e.g. PBST, macroporoushydrophobic cloths incubated with an antigen-conjugate mixture fail toproduce the same intensity of colours (upon incubation inABTS-indicator) as macroporous hydrophobic cloths incubated with acontrol mixture consisting of the conjugate in the absence of freeantigen.

While it is not desired to be bound by any theory, it is believed thatantigen present in the test sample combines with the conjugate, thuspreventing its interaction with the antigen-treated cloth. In thismanner, the presence of antigen in a test sample will diminish theamount of colour produced in the test, while the control sample (minusfree antigen) gives proof of the functional integrity of the conjugate.

The detection of B. abortus antigen LPS, using the enzyme immunoassaywith LPS-coated macroporous polyester cloth has thus been provided asanother embodiment of this invention. The enzyme immunoassay, whichrequires only one incubation with the immunological reagent, provides aninternal control for the quality of the reagent used.

Before describing various embodiments of this invention, a descriptionof the reagents used will be given. Chemicals used were of theanalytical reagent grade. All biochemicals were purchased from SigmaChemical Co. Distilled water (H₂ O) was employed as a universal solvent.Antigens and bovine antisera were provided by the Animal DiseasesResearch Institute (ADRI) in Nepean, Ontario, Canada. Some materialsemployed as solid phases (i.e. cellulose cotton and nylon cloths) werecommercially available as generic materials and were acquired locally,in the Ottawa, Canada area, whereas non-woven polypropylene filter clothwas commercially available as a generic product and was purchased fromAldrich Chemical Co. and a variety of polyester cloths were commerciallyavailable under the trademark SONTARA and were obtained from DuPont.

Enzyme Immunoassay Reagents

    ______________________________________                                        (1) 0.06M carbonate buffer (pH 9.6)                                               NaHCO.sub.3                3.8    g                                           Na.sub.2 CO.sub.3          1.93   g                                           Add H.sub.2 O and NaOH (if necessary) to 1,000 ml.                        (2) 0.01M phosphate-buffered saline (pH 7.2) (PBS)                                NaH.sub.2 PO.sub.4.2H.sub.2 O                                                                            0.31   g                                           Na.sub.2 HPO.sub.4         1.1    g                                           NaCl                       8.5    g                                       (3) PBS with TWEEN 20 (PBST)                                                      PBS                        1000   m                                           TWEEN 20 [TWEEN 20 is the registered                                                                     0.5    ml                                          Trade Mark of an emulsifier                                                   comprising polyoxyethylene                                                    derivatives of fatty acid                                                     partial esters of sorbitol                                                    anhydrides of Atlas Chemical                                                  Industries]                                                               (4) Indicator system for horseradish peroxidase                                   (ABTS-indicator)                                                              0.05M citrate buffer (pH 4.5)                                                                            20     ml                                          40 nM 2,2'-Azino-di-(3-ethylbenzthiazoline                                                               0.5    ml                                          sulfonic acid) (ABTS)                                                         0.5M H.sub.2 O.sub.2       0.02   ml                                      (5) Indicator system for alkaline phosphatase                                     diethanolamine             2.62   g                                           P-nitrophenyl phosphate    .025   g                                       ______________________________________                                    

Add H₂ O and HCl to 25 ml to obtain a final pH 9.8.

Preparation of Affinity Purified Anti-Brucella Antibodies from BovineAntiserum

Anti-Brucella antibodies were purified from bovine antiserum by theaffinity purification method. It is based on the adsorption ofanti-Brucella antibodies onto the antigenic surface of whole killed B.abortus cells mixed with antiserum. Cells with adsorbed antibodies canthen be separated from the serum by centrifugation, and the antibodiescan be recovered by exposure to a low pH environment with subsequentremoval of the cells by centrifugation. The method is simple to perform,inexpensive, and usually results in high yields of specific antibodies.

Ten milliliters of standard plate test antigen, consisting of wholeheat-killed B. abortus cells (strain 413, biotype 1) suspended inphenol-saline (i.e., 0.85% NaCl and 0.5% phenol in H₂ O) at aconcentration of 4×10" cells/ml, were dispensed in a 50 ml-capacityround bottom polycarbonate centrifuge tube. The cells were pelleted bycentrifugation at 10,000×g, for 10 minutes. The pellet was then washedtwice in 0.1M glycine-HCl (pH 2.24) to remove any acid-soluble materialpresent on the cell surface, followed by two washings in 0.1M Tris-HClbuffer (pH 7.0). Care was taken to disperse the cells as gently aspossible during resuspension (a glass stirring rod is convenient forthis purpose). To the final washed pellet was added 25 ml of bovineantiserum, in which the cells were dispersed. The suspension was allowedto stand at room temperature for 30-40 minutes, with gentle stirringevery 5-10 minutes. The suspension was then centrifuged as above and thesupernatant discarded. The resulting pellet was washed three times in 25ml of 0.1M Tris-HCl buffer (pH 7.0) to remove any loosely adsorbedmaterial. Brucella-specific antibody was recovered by resuspending thefinal washed pellet in 25 ml of 0.1M glycine-HCl (pH 2.24). The cellswere immediately removed by centrifugation and antibody-rich supernatantwas transferred to a vessel containing 10 ml of 1.0M Tris-HCl buffer (pH8.0) in order to abrogate the harsh low pH environment. The remainingcell pellet was processed in this manner a second time to improveantibody recovery, and the final supernatants were pooled. The antibodysolution was then dialyzed against PBS for 24 hours at 4° C., with atleast three changes of buffer. Precipitate material arising in thedialysate was stored at -80° C. until use. Whenever necessary, theprotein in the dialysate was concentrated using an Amicon proteinconcentrator.

Unless otherwise specified, antiserum used in the Examples was serumobtained from chronically infected cattle which have high titers ofanti-Brucella antibody. B. abortus cells used were heat-killed standardplate test antigen (whole cells) which is strain 413, biotype 1,suspended in phenol-saline.

Preparation of an Enzyme-antibody Conjugate by the Periodate OxidationMethod

A modified version of the method developed by Nakane and Kawaoi wasemployed for conjugating horseradish peroxidase with anti-Brucellaantibody. For this, highly purified horseradish peroxidase (RZ=3) wasused, where RZ is the light absorbance ratio of A₄₀₃ /A₂₇₅ for a givenenzyme solution. Five milligrams of horseradish peroxidase (Sigma, typeVI, RZ=3.0) were dissolved in a mixture of 1.0 ml of 0.3M sodiumcarbonate (pH 8.1) and 1.0 ml of 0.08M NaIO₄. The solution was mixedgently for 30 minutes at room temperature, and 1.0 ml of 0.16M ethyleneglycol was then added. This was further mixed at room temperature for 1hour. The latter solution was then dialyzed against 5 l of 0.01M sodiumcarbonate buffer (pH9.5) at 4° C. for 16-24 hours. To this dialysate wasadded 0.5 ml of PBS containing ca. 5 mg of affinity purifiedanti-Brucella antibody and 0.5 ml of 1.01M carbonate buffer (pH 9.5).The solution was incubated for 3 hours at room temperature, with gentlemixing. At the end of the incubation, 5 mg of NaBH₄ were added to thesolution which was then left to stand at 4° C. for 3 hours. The solutionwas then dialyzed extensively against PBS at 4° C. Any precipitablematerial formed in the dialysate was removed by centrifuging for 10minutes in an eppendorf microfuge. The resulting supernatant constitutedthe stock conjugate, which was designated conjugate, and was stored at-20° C. until use.

Collection of Antiserum

An adult cow suffering from chronic brucellosis was bled at one-weekintervals over a period of approximately three weeks. At each bleeding,ca. 1 l of blood was collected in glass bottles and immediatelyincubated at 37° for 2 hours, followed by overnight storage at 4° C. toallow for complete clot formation. The serous liquid was decanted intoseparate vessel and centrifuged at 8,000×g to remove any remaining bloodcells. The supernatant thus obtained was stored at -20° C.

A large stock of antiserum, which was used as a source of antibody forall subsequent experiments, was prepared by pooling all serum samplesobtained after the prescribed succession of bleedings, and was stored at-80° C. until use.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings,

FIG. 1 is a graph showing the effect of varying the treatingconcentration on the signal generated in the polypropylene cloth-basedcloth enzyme immunoassay;

FIG. 2 is a graph showing the partial denaturation of bovine antiserumantibodies by audification;

FIG. 3 is a graph showing the partial denaturation of bovine antiserumand affinity purified antibodies by heat; and

FIG. 4 is a graph showing diluted antiserum as a source of coatingantibody.

DESCRIPTION OF PREFERRED EMBODIMENTS

The following are Examples of this invention:

I(a) Determination of the Optimum Affinity Purified Coating AntibodyConcentration for Enzyme Immunoassay Using Macroporous Polypropylenecloth

Macroporous polypropylene filter cloth pieces (6×6 mm) were coated with50 μl of various concentrations of affinity purified anti-Brucellaantibody per piece (a concentration range of 0.2 to 1.0 mg of protein/mlwas chosen). After the required overnight incubation period, the clothswere washed with PBST and subsequently incubated with 30 μl of B.abortus cells (strain 413, biotype 1) diluted to 4×10⁸ cells/ml in PBS,for 30 minutes at room temperature. A series of negative control cloths(incubated without antigen) was also included. The macroporoushydrophobic cloths were then washed with PBST and probed with 25 μl ofconjugate diluted 1:1,000 in PBST, and incubated for 30 minutes at roomtemperature. After washing with PBST, the macroporous hydrophobic clothswere assayed for retained horseradish peroxidase activity by immersionin 0.5 ml of ABTS-indicator solution for 30 minutes, at roomtemperature, and the reaction was stopped by addition of 0.5 ml of 0.1MNaF. Absorbance was read at 650 nm.

As shown in FIG. 1, the assay response peaks at a concentration of 0.2mg/ml.

I(b) Pretreatment (Partial Denaturation) of the Coating Antibody

The use of antiserum for the direct coating of the macroporoushydrophobic cloth was investigated as an effective and economicalalternative to purified antibody.

In order to achieve maximum immobilization of the antibodies present inthe antiserum, a simple procedure was developed for the pretreatment ofthe coating antiserum to incur partial denaturation of the antibodies,thus rendering them more capable of interacting with the macroporoushydrophobic cloth surface, to the exclusion of other serum proteinswhich might compete for binding sites on the cloth. In order to improvethe detectability of the enzyme immunoassay employing bovine antiserumas the source of coating antibody, partial denaturation procedures weredeveloped using an acidic pH and heat. The following describes aninvestigation undertaken to determine the optimum time of exposure to apH 2.5 environment for the improved immobilization of antibodies fromantiserum on macroporous polypropylene cloth.

Three separate 0.85 ml aliquots of antiserum were mixed with 0.3 ml eachof 0.4M glycine-HCl buffer (pH 1.5) to produce a final pH 2.5. Azero-time exposure sample consisting of 0.85 ml of antiserum plus 0.6 mlof 1.0M Tris-HCl buffer (pH 7.0) was also prepared as a control. Eachacidified sample was allowed to stand at room temperature for either 5,10, or 20 minutes, after which they were immediately neutralized byaddition of 0.3 ml of 1.0M Tris-HCl (pH 8.0). These samples were thenused to coat macroporous polypropylene filter cloth pieces. The effectof time of exposure to pH 2.5 on the cloth enzyme immunoassay signalgenerated is presented in FIG. 2.

FIG. 2 shows that an approximate five-fold improvement in the assay'ssensitivity was achieved by exposure of the antiserum to pH 2.5 for 10minutes.

In a second experiment, the effect of exposing bovine antiserum andaffinity purified antibodies to heat on their abilities to serve assources of coating antibodies was examined. One milliliter samples ofantiserum and the affinity purified antibody dialysate (containing 0.27mg of protein/ml) were incubated for 10 minutes at either 25°, 65°, 70°,75°, or 80° C. These were then allowed to cool to room temperature andused to coat macroporous polypropylene cloths as previously described.The enzyme immunoassay protocol employed was the same as in the previousexperiment, with the exception that the antibody-coated macroporoushydrophobic cloths were incubated with 30 μl of B. abortus plate testantigen diluted to 4×10⁷ cells/ml in PBS. The results of this experimentare presented in FIG. 3, where the signal generated in the enzymeimmunoassay is plotted against temperature for both antiserum andaffinity-purified antibody.

FIG. 3 shows that an even greater improvement in sensitivity wasobtained by heating the serum at 75° C. for 10 minutes. The sensitivityof the enzyme immunoassay employing affinity purified antibody remainedessentially unchanged over the range of temperatures tested.

These experiments have demonstrated the usefulness of either exposure toa low pH or heat in improving the sensitivity of the assay usingantiserum as the source of coating antibody.

Without wishing to be bound by theory, it is believed that theimprovement in the sensitivity of the antiserum-based immunoassay is aconsequence of an increase in the hydrophobicity of the denatured Fcregion of the antibodies, which in turn causes these to be adsorbed morestrongly to the hydrophobic cloth surface and in greater numbers. Thepartially denatured state may also ensure that the antibodies adhere tothe solid phase in a more ideal orientation, with the Fc region affixedto the cloth surface and the Fab segments free to interact with theantigen. Treating the affinity-purified antibodies with heat did notseem to confer any particular advantage. Thus, these affinity-purifiedantibodies cannot be beneficially altered further by heat treatment.

I(c) Diluted Bovine Antiserum as a Source of Coating Antibody

The feasibility of applying diluted antiserum in the enzyme immunoassaywas investigated in the following experiment.

Aliquots of bovine antiserum were diluted 2, 4, 6, 8, and 10 times inPBS. The diluted samples were partially denatured by heating at 75° C.for 10 minutes. These were then cooled to room temperature and appliedto 6×6 mm macroporous polypropylene filter cloth pieces, which weresubsequently employed in the enzyme immunoassay according to theprocedure used in the previous example. The results are presented inFIG. 4, where the enzyme immunoassay signal generated is plotted againstthe serum dilution factor.

FIG. 4 shows that there was no appreciable decline in the sensitivity ofthe assay throughout the range of coating serum dilutions examined.Therefore, it was concluded that bovine antiserum diluted 1:10 in PBS,with subsequent heating at 75° C. for 10 minutes, can serve as asuitable source of coating antibody in the enzyme immunoassay. Theprecise dilution factor used for a given batch of antiserum will, ofcourse, depend on the specific antibody titer of that serum.

(II) Detectability of the Enzyme Immunoassay For Brucella Antigens

The detectability of Brucella cells and lipopolysaccharide antigens(LPS) by the macroporous polypropylene enzyme immunoassay was examinedin this Example. Unless otherwise stated, all antibody-coated clothsused in the following experiments were prepared with bovine antiserumdiluted 1:lo in PBS and heated at 75° C. for 10 minutes.

II(a) Detectability of the Whole Cell Assay Employing Antibody CoatedMacroporous Polypropylene Filter cloth

Antibody-coated macroporous polypropylene filter cloth pieces wereincubated for 30 minutes at room temperature with 30 μl of B. abortuscell suspensions containing either 1.2×10⁵, 1.2×10⁴, or 1.2×10³, cellsin PBS. Each macroporous hydrophobic cloth in this series of experimentswas prepared in quadruplicate, as were macroporous hydrophobic cloths towhich 30 μl of PBS containing no antigen was added. These were thenwashed with PBST and probed with 25 μl of conjugate diluted 1:1000 inPBST, for 30 minutes at room temperature. At the end of this period, themacroporous hydrophobic cloths were washed with PBST and assayed forretained horseradish peroxidase by immersion in ABTS-indicator solutionfor 3 hours, in order to optimize the final enzyme signal. The enzymereaction was stopped by addition of 0.5 ml of 0.1M NaF. Table 1 showsthe relationship between the quantity of antigen added and thecorresponding cloth enzyme immunoassay signal obtained.

                  TABLE 1                                                         ______________________________________                                        Detectability of the Whole Cell Assay Employing                               Antibody-Coated Macroporous Polypropylene Filter                              Cloth                                                                         No. of Cells Applied                                                                         A.sub.650                                                      Per Cloth      1      2         3    4                                        ______________________________________                                        1.2 × 10.sup.5                                                                         0.186  0.213     0.234                                                                              0.245                                    1.2 × 10.sup.4                                                                         0.107  0.080     0.106                                                                              0.075                                    1.2 × 10.sup.3                                                                         0.054  0.050     0.060                                                                              0.055                                    0              0.060  0.051     0.058                                                                              0.055                                    ______________________________________                                    

Table 1 shows that the macroporous polypropylene cloth immunoassay candetect 10⁴ cells using a 36 mm² polypropylene cloth.

II(b) Detectability of Brucella LPS by Enzyme Immunoassay UsingMacroporous Polypropylene Cloth

The assay procedure employed for the detection of whole cells usingantibody-coated macroporous polypropylene filter cloth was applied tothe detection of B. abortus LPS. In this Example, antibody-coatedmacroporous hydrophobic cloths were incubated for 30 minutes at roomtemperature with 30 μl of PBS containing either 3, 0.3, or 0.03 ng ofLPS, or PBS alone. Each macroporous hydrophobic cloth was prepared inquadruplicate. These were then processed in the immunoassay aspreviously described. The results are presented in Table 2.

                  TABLE 2                                                         ______________________________________                                        Detectability of LPS by Enzyme Immunoassay Using                              Macroporous Polypropylene Cloth                                               LPS Applied   A.sub.650                                                       per Cloth (ng)                                                                              1      2         3    4                                         ______________________________________                                        3.00          1.350  1.302     1.440                                                                              1.100                                     0.30          0.261  0.225     0.216                                                                              0.230                                     0.03          0.063  0.058     0.066                                                                              0.068                                     0             0.068  0.040     0.043                                                                              0.056                                     ______________________________________                                    

Table 2 shows that the detectability of this enzyme immunoassay occurredat approximately 0.3 ng (or 300 picograms) of LPS applied permacroporous hydrophobic cloth piece.

II(c) Performance of the Enzyme Immunoassay Under Simulated ClinicalConditions

In the routine diagnosis of brucellosis, Brucella organisms are oftenrecovered from infected animals in milk; vaginal secretions;supramammary, retropharyngeal, internal iliac, and lumbar lymph nodes;spleen tissue; uterine tissue; and in some instances, blood. As thesematerials constitute complex environments for the detection of antigens,it was determined whether or not undefined sample components might beprohibitive to antigen detection by the enzyme immunoassay. Anotheraspect of clinical specimens examined was the interference ofanti-Brucella antibodies present in the samples to the antigen assay bythe enzyme immunoassay. The ability of the macroporous polypropylenefilter cloth-based enzyme immunoassay to detect B. abortus antigens inbody fluids and tissue homogenates of bovine origin was examined inExample II. Antibody-coated cloth was prepared as in the previousExample.

II(d) Detection of B. Abortus in Bovine Body Fluids and TissueHomogenates

In order to ascertain the ability of the enzyme immunoassay to detect B.abortus antigens in simulated clinical specimens, tissues obtained froma cow which was a serological reactor for B. abortus, but culturenegative, were artificially innoculated with whole cells and assayed forthe presence of antigen as described below. The presence of endogenouscirculating antibody specific for B. abortus offered an opportunity toassess the performance of the assay under conditions which mighttheoretically interfere with the capture of antigen in such samples. Thepossible inhibitory effect of endogenous antibody was alleviated bysubjecting the test samples to extreme heat (3 hours, 70° C.) prior toperforming the assay.

Tissue homogenates were prepared by homogenizing in a stomacher andadding sufficient PBS to produce a fluid consistency (approximately 0.5ml per gram of tissue). Homogenates were made from the following bovinetissues; inguinal lymph nodes, spleen, and uterine horn.

A sample from each homogenate was inoculated with sufficient B. abortuscells to give a final concentration of 8×10⁵ cells/ml Milk and serumsamples from a healthy animal were likewise inoculated. These samples,along with their uninoculated counterparts, were heated at 70° C. for 3hours, after which they were cooled to room temperature. Antibody-coatedmacroporous polypropylene filter cloths were then added to triplicatetest tubes containing 0.5 ml of heated sample (one macroporoushydrophobic cloth/tube) and incubated for 30 minutes at room temperaturewith constant gentle shaking. The macroporous hydrophobic cloths werethen removed and processed in the enzyme immunoassay as previouslydescribed. Macroporous hydrophobic cloths were assayed for retainedconjugate by immersion in 0.5 ml of ABTS-indicator solution for 30minutes. The results are shown below in Table 3.

                  TABLE 3                                                         ______________________________________                                        Detection of Antigens Suspended in Body Fluids and Tissue                     Homogenates.                                                                          A.sub.650                                                                    Specific.sup.b Control.sup.c                                           Sample.sup.a                                                                           1        2      3      1    2      3                                 ______________________________________                                        A        0.185    0.240  0.224  0.070                                                                              0.090  0.083                             B        0.164    0.177  0.170  0.057                                                                              0.060  0.061                             C        0.132    0.170  0.162  0.022                                                                              0.027  0.020                             D        0.159    0.165  0.162  0.020                                                                              0.030  0.020                             E        0.175    0.192  0.195  0.045                                                                              0.042  0.040                             ______________________________________                                         .sup.a Tissue homogenates from spleen (A), inguinal lymph nodes (B),          unterine hore (C), plus, normal bovine serum (D) and normal bovine milk       (E).                                                                          .sup.b Cloths incubated with inoculated samples.                              .sup.c Cloths incubated with uninoculated samples.                       

Table 3 demonstrates the assay response for each sample tested In mostcases, the control signals (i.e., those obtained from the uninoculatedsamples) remained low, whereas those arising from the correspondinginoculated samples were distinct. The experiment demonstrates theability of the enzyme immunoassay to detect B. abortus antigenssuspended in various biological samples.

III Detection of Bovine Viral Diarrhea (BVD) Antigen

The applicability of the ability of the enzyme immunoassay to detectantigens of bacterial origin to the detection of viral antigens will nowbe described in this Example.

Bovine viral diarrhea is an enteric disease posing a serious threat toexposed livestock. The etiological agent is a virus which is referred toas bovine viral diarrhea (BVD) antigen. At present, an enzymeimmunoassay method is not used for the detection of BVD antigen inclinical specimens.

The strategy employed for the BVD assay was similar to that usedpreviously for the detection of Brucella antigens macroporouspolypropylene filter cloth was coated with anti-BVD antibodies andsubsequently incubated with BVD antigen, and the captured material wasdetected by probing with an enzyme-antibody conjugate (prepared bycoupling antibodies from anti-BVD antiserum to horseradish peroxidase bythe periodate oxidation method). Whole antiserum was employed in orderto make the required conjugate. The feasibility of using whole antiserumfor this purpose was first tested by conjugating whole anti-Brucellaantiserum with horseradish peroxidase and applying the resultingconjugate in the B. abortus enzyme immunoassay, thus affording anopportunity to compare the performance of such a conjugate with anestablished system.

III(a) Synthesis and Testing of a Brucella-specific Enzyme-AntibodyConjugate Made With Whole Bovine Antiserum

Whole bovine anti-Brucella antiserum was conjugated to horseradishperoxidase according to the periodate oxidation method: 0.5 ml of wholeserum was dialyzed overnight at 4° C. against 0.01M sodium carbonatebuffer (pH 9.5). The resulting dialysate was mixed with 5 mg ofperiodate-activated horseradish peroxidase and incubated for 2.5 hoursat room temperature. Sodium borohydride (NaBH₄) was then added asprescribed, and incubated for 3 hours at 4° C. against PBS, and thefinal dialysate was microfuged (10 minutes, at 4° C.). The cleareddialysate constituted the conjugate stock.

The performance of this conjugate in the immunoassay was compared tothat of the conjugate prepared with purified antibody. Two series ofBrucella-specific antibody-coated macroporous polypropylene filter clothpieces were incubated with 30 μl of B. abortus cells diluted to 4×10⁸4×10⁷, or 4×10⁶ cells/ml in PBS, for 30 minutes at room temperature. Themacroporous hydrophobic cloths were then washed in PBST, and one serieswas probed with 25 μl of the conjugate prepared with affinity purifiedantibody diluted 1:100 in PBST while the other series was probed with 25μl of the conjugate prepared with whole antiserum diluted 1:100 in PBST.These were incubated for 30 minutes, at room temperature, andsubsequently washed with PBST. The macroporous hydrophobic cloths wereassayed by immersion in 0.5 ml of ABTS-indicator solution for 30 min.The results are shown below in Table 4.

                  TABLE 4                                                         ______________________________________                                        Relative Performance of Conjugate D in the Enzyme                             Immunoassay.                                                                                A.sub.650                                                       No. of Cells Applied                                                                          conjugate.sup.1 *                                                                              conjugate.sup.2 **                           per Cloth       1      2         1    2                                       ______________________________________                                        1.2 × 10.sup.7                                                                          0.852  0.932     0.631                                                                              0.598                                   1.2 × 10.sup.6                                                                          0.385  0.400     0.153                                                                              0.180                                   1.2 × 10.sup.5                                                                          0.081  0.092     0.081                                                                              0.080                                   0               0.010  0.012     0.070                                                                              0.075                                   ______________________________________                                         *prepared by coupling affinity purified antibody to HRP                       **prepared by coupling whole antiserum to HRP                            

According to Table 4, the response of the enzyme immunoassay usingconjugate² was somewhat dampened throughout the series of antigendilutions tested, as compared to the performance of conjugate¹ in theassay. However, the Example demonstrates the feasibility of employingsuch a conjugate.

III(b) Adapted Enzyme Immunoassay for the Detection of BVD Antigen

A BVD-specific conjugate was prepared by coupling whole anti-BVDantiserum to horseradish peroxidase, in order to adapt the enzymeimmunoassay for the direct detection of BVD antigen.

A sample of anti-BVD antiserum (precise titer unknown) was obtained froma cow vaccinated with killed BVD virus. An anti-BVD serumprotein-horseradish peroxidase conjugate was prepared by coupling 0.5 mlof anti-BVD antiserum dialyzed against sodium carbonate buffer to 5 mgof horseradish peroxidase as in the previous Example. The resultingproduct was designated conjugate³.

The standard antigen employed in the immunoassay originated from acommercial BVD vaccine, consisting of killed whole virus suspended insaline containing an unknown quantity of bovine serum albumin (added asa stabilizer) and a variety of anti-microbial agents. The quantity ofviral antigen per unit volume of the vaccine was not disclosed by themanufacturer. The vaccine was dialyzed overnight at 4° C. against PBSprior to use.

Macroporous polypropylene filter cloth pieces were coated with 60 μl ofpartially denatured anti-BVD antiserum (i.e., heated at 75° C. for 10minutes) and incubated at room temperature as previously described.Antibody-coated macroporous hydrophobic cloths were incubated for 30minutes at room temperature with 30 μl of dialyzed BVD vaccine dilutedeither 1 10, 1:100 or 1:1,000 in PBS. Undiluted vaccine and PBS alonewere also included in the series, and each macroporous hydrophobic clothwas prepared in quadruplicate. After the incubation period, themacroporous hydrophobic cloths were washed with PBST and probed with 25μl of conjugate³ diluted 1:100 in PBST, as previously. These were thanassayed by immersion in 0.5 ml of ABTS-indicator solution for 30minutes. The results are shown below in Table 5.

                  TABLE 5                                                         ______________________________________                                        Detection of BVD Antigen by the Enzyme Immunoassay                                         A.sub.650                                                        Antigen Dilution                                                                             1      2         3    4                                        ______________________________________                                        undiluted      1.230  1.190     1.090                                                                              1.200                                    1:10           0.246  0.280     0.265                                                                              0.210                                    1:100          0.091  0.110     0.115                                                                              0.095                                    1:1000         0.060  0.071     0.055                                                                              0.050                                    no antigen     0.055  0.053     0.051                                                                              0.042                                    ______________________________________                                    

Table 5 shows that the detectability of the BVD assay, in the formtested, was fixed somewhere in the range of 1:10 to 1:100 dilution ofthe dialyzed vaccine. These results clearly demonstrate the ability ofthe immunoassay to detect BVD antigen.

II(c) Alternative Solid Phases for Use in the Enzyme Immunoassay

A series of Examples was carried out to determine the usefulness ofother materials as solid phases for capture and detection of B. abortuscells.

Macroporous polypropylene filter cloth was compared to a variety ofother materials employed as solid phases in the immunoassay. Thematerials tested were macroporous 100% nylon cloth (acquired locally),in the Ottawa, Canada area, macroporous woven polyester cloth (acquiredlocally), in the Ottawa, Canada areas, macroporous nonwoven polyestercloth (DuPont), cellulose acetate membrane (Gelman), cellulose nitratemembrane (Schleicher and Schuell), analytical paper (Schleicher andSchuell), and a macroporous polyethylene filter (1.5 mm thickness,supplier unknown). These were all cut into 6×6 mm square pieces, andcoated with 60 μl of partially denatured bovine anti-Brucella antiserumdiluted 1:10 in PBS, as previously. The materials were then incubatedwith 30 μl of B. abortus cells diluted to 4×10⁷ cells/ml in PBS for 30minutes at room temperature. A parallel series to which PBS containingno antigen was added was also included, and each solid phase wasprepared in duplicate. After the incubation period, the materials werewashed with PBST and probed with conjugate (prepared with affinitypurified antibody) as usual. These were then assayed by immersion in 0.5ml of ABTS-indicator solution for 30 minutes. The performance of eachsolid phase in the assay is shown below in Table 6.

                  TABLE 6                                                         ______________________________________                                        Performances of Various Solid Phases Employed in the                          Enzyme Immunoassay                                                                            A.sub.650                                                                     specific.sup.a                                                                              control.sup.b                                   Solid Phase     1         2      1      2                                     ______________________________________                                        Polypropylene filter cloth                                                                    0.428     0.430  0.012  0.010                                 Nylon cloth     0.320     0.338  0.011  0.009                                 Woven polyester cloth                                                                         0.840     0.860  0.035  0.026                                 Nonwoven polyester cloth.sup.c                                                                0.810     0.792  0.033  0.020                                 Cellulose acetate membrane                                                                    0.220     0.186  0.100  0.122                                 Cellulose nitrate membrane                                                                    0.282     0.235  0.185  0.192                                 Polyethylene filter                                                                           0.450     0.455  0.061  0.072                                 Analytical paper                                                                              0.416     0.422  0.400  0.421                                 ______________________________________                                         .sup.a Cloths incubated with antigen                                          .sup.b Cloths incubated without antigen                                       .sup.c A variety of DuPont polyester cloths were examined and the results     with these cloths were similar to data shown here                        

Table 6 shows that of the solid phases tested, the highest signals wereobtained with the macroporous woven polyester and macroporous nonwovenpolyester cloths. Other materials that are useful in the enzymeimmunoassay are macroporous nylon cloth and the macroporous polyethylenefilter, which produce similar results to those obtained usingmacroporous polypropylene filter cloth.

The main advantage of macroporous hydrophobic cloths as supports inenzyme immunoassay is that they provide a large volume for absorptionper surface area for antigen-antibody interaction. Macroporouspolyethylene, macroporous nylon, and macroporous polyester cloths, byvirtue of their hydrophobic characteristics, have been found to adsorband absorb antibodies and thus provided a large surface area for antigencapture. Other materials amenable to the enzyme immunoassay conceptshould have included cellulose acetate and cellulose nitrate membranesand filter paper. However as the results above indicated, they are notuseful according to the present invention.

IV Comparison of the Enzyme Immunoassay Using Macroporous PolypropyleneFilter Cloth, a Flat Polypropylene Sheet and a Polystyrene MicrotiterPlate Surface

The enzyme immunoassay response arising from the use of anantibody-coated plastic polypropylene sheet and a polystyrene microtitersurface, which have very limited surface areas available for antibodyadsorption, and hence antigen capture, to the assay response obtainedusing macroporous hydrophobic polypropylene filter cloth when variousquantities of antigen are applied was compared.

A flat polypropylene sheet, cut into 6×6 mm pieces, was coated withanti-Brucella antibodies as were 6×6 mm pieces of polypropylene filtercloth. Several wells in a polystyrene microtiter plate were also coated.These were then incubated with 30 μl of PBS containing either 1.2×10⁷,1.2×10⁶, 1.2×10⁵, 1.2 ×10⁴, B. abortus cells (plate test antigen), orPBS alone, for 30 minutes at room temperature. The materials weresubsequently processed in the enzyme immunoassay as in the previousExample, with the exception that enzyme activity was assayed byimmersion in 0.5 ml of ABTS-indicator solution for 3 hours. Eachdetermination was done in triplicate, and the results are shown below inTable 7.

                  TABLE 7                                                         ______________________________________                                        Detectability of the Enzyme Immunassay Employing                              Three Different Solid Phases.                                                         No. of Cells Applied Per Piece                                        A.sub.650 1.2 × 10.sup.7                                                                   1.2 × 10.sup.6                                                                   1.2 × 10.sup.5                                                                 1.2 × 10.sup.4                                                                 0                                   ______________________________________                                        Polypropylene                                                                           5.02     1.02     0.29   0.09   0.04                                Filter 2  5.12     1.24     0.29   0.08   0.03                                Cloth 3   5.30     1.15     0.24   0.08   0.03                                Plastic 1 1.02     0.43     0.06   0.03   0.04                                Polypropylene                                                                           1.01     0.32     0.07   0.02   0.04                                2                                                                             Sheet 3   0.96     0.87     0.07   0.04   0.03                                Polystyrene 1                                                                           1.08     0.28     0.08   0.02   0.03                                Microtiter Plate                                                                        1.22     0.35     0.09   0.03   0.04                                2                                                                             Surface 3 1.01     0.30     0.08   0.04   0.02                                ______________________________________                                    

Table 7 shows that the successful detection of very small quantities ofantigen must require a significantly large capturing surface in order toincrease the probability of interaction between the solid phase and theantigen during the limited incubation period involved. This expectationis confirmed by the results obtained using an antibody-coated plasticpolypropylene sheet and a polystyrene microtiter plate surface as solidphases, which failed to detect small quantities of antigen to which themacroporous hydrophobic polypropylene cloth responded, and which showeda greatly diminished sensitivity throughout the range of antigenconcentration tested.

V Commercial Adaptation of the Enzyme Immunoassay: Dipstick ofHydrophobic Cloth

A commercial form of the enzyme immunoassay was developed forapplication of the enzyme immunoassay in any number of circumstances(e.g., diagnostic laboratory and field testing, etc.). One practicalform consists of affixing a small rectangular piece of macroporouspolypropylene filter cloth to a strip, e.g. of cellulose acetate, whichallows for the easy retrieval of the antibody-coated macroporoushydrophobic cloth from test samples and provides a convenient means ofhandling the macroporous hydrophobic cloth throughout the assayprocedure. It is necessary to ensure that the bond created between themacroporous polypropylene cloth and the cellulose acetate does not alterthe properties of the former or result in any structural features of themacroporous hydrophobic cloth/strip junction which might causenon-specific retention of the conjugate.

A bond was created by first dissolving one edge of a cellulose acetatestrip having the dimensions 21/2×1/4 macroporous polypropylene clothpiece of the same thickness, making sure not to allow any overlapping ofone edge over the other. Upon evaporation of the acetone, a strong bondwas formed between the cellulose acetate strip and the macroporouspolypropylene filter cloth piece. The cloth portion of the resultingtest strip was coated with antibody by applying 100 μl of partiallydenatured bovine anti-Brucella antiserum diluted 1:10 in PBS andincubating overnight at room temperature, followed by washing with PBSTas previously described. The antibody-coated test strip was tested inthe enzyme immunoassay in the manner described below.

Test strips were incubated with either 30 μl of PBS containing 1.2×10⁶B. abortus cells (plate test antigen) or 30 μl of PBS alone, for 30minutes at room temperature. These were then washed with PBST andincubated for 30 minutes at room temperature with 25 μl of conjugate¹diluted 1:1,000 in PBST. The macroporous hydrophobic cloth portions ofthe test strips were then washed with PBST, and were subsequentlyassayed for retained enzyme activity by immersion in 1 ml ABTS-indicatorsolution for 30 minutes with gentle shaking. The reaction was stopped byaddition of 0.5 ml of 0.1M NaF and absorbance was read at 650 nm. Eachdetermination was performed in quadruplicate, and the results of theassay are shown below in Table 8.

                  TABLE 8                                                         ______________________________________                                        Application of Antibody-Coated Test Strips in Enzyme                          Immunoassay                                                                   No. of Cells Applied                                                                         A.sub.650                                                      per Test Strip 1      2         3    4                                        ______________________________________                                        1.2 × 10.sup.6                                                                         0.411  0.427     0.415                                                                              0.435                                    0              0.023  0.016     0.020                                                                              0.021                                    ______________________________________                                    

The results of the enzyme immunoassay demonstrate the ability of theantibody-coated test strips to detect B. abortus antigens at theconcentration tested. The background level of enzyme activity wasnegligible, thus satisfying one of the important requirements of theimmunoassay. These results were reproducible.

V Application of a Macroporous Hydrophobic Cloth as an Adsorbant andAbsorbant of Antigen in Enzyme Immunoassay for B. Abortus LPS.

The following experiment shows the application of macroporoushydrophobic cloth as an adsorbent and absorbent of antigen for theimmunoassay.

Macroporous polyester non-woven cloths (DuPont SONTARA 8100™) wereincubated with 50 μl per 6×6 mm piece of 2 μg/ml solution of B. abortusLPS in PBS, overnight at room temperature.

The macroporous hydrophobic cloths were then washed with PBST. Eachcloth was incubated for 30 min., at room temperature, with 25 μl ofeither of the following preparations: 50 μl of PBS containing 90 ng ofB. abortus LPS plus 25 μl of antibody-horseradish peroxidase conjugatediluted 10,000× in PBST, 50 μl of PBS containing a ng of 1PS plus 25 μlof diluted conjugate, 50 μl of PBS containing 0.9 ng of LPS plus 25 μlof diluted conjugate, or 50 μl of PBS alone plus 25 μl of dilutedconjugate.

The macroporous hydrophobic cloths were then washed with PBST andassayed for HRP by immersion in 0.5 ml of ABTS-indicator for 90 min., atroom temperature. The reaction was stopped by addition of 0.5 ml of 0.1MNaF, and absorbance were read at 650 nm. The results of this experimentare recorded in Table 9 below.

                  TABLE 9                                                         ______________________________________                                        Enzyme Immunoassay for the Detection of B. Abortus                            LPS Using Macroporous Polyester Non-Woven Cloths                              LPS (ng)    A.sub.650                                                         applied per cloth                                                                        1         2       Visual observation                               ______________________________________                                        30         0.02      0.01    yes                                              3          0.127     0.115   yes                                              0.3        0.22      0.20    no                                               0          0.20      0.23    --                                               ______________________________________                                    

The results show clearly that at least 30 ng and 3 ng of LPS weredetectable by simple visual examination, where the corresponding samplesproduced less colour than the control. However, it is equally importantto note that the enzyme immunoassay might successfully detect even lowerquantities of LPS by colorimetric measurement. The enzyme immunoassayoffers the distinct advantages of providing an internal control forconjugate quality and requiring only one incubation step for thedecisive immunological reaction. Such advantages make the enzymeimmunoassay more easily adaptable for field testing.

It is believed that the detectability limit of this enzyme immunoassaymay be improved by coating the macroporous polyester cloth with alimited quantity of antigen, making the free antigen present in the testsample more competitive for the conjugate.

An improved enzyme-antibody conjugate having a higher specific activityhas also been provided which should allow for the use of highly dilutedconjugate in the assay, thus describing the quantity of free antigenrequired to prevent attachment of the conjugate to the macroporoushydrophobic cloth (thus increasing the sensitivity of antigendetection).

GENERAL OBSERVATIONS

The enzyme immunoassay device of this invention thus employs macroporoushydrophobic cloths as surfaces. Macroporous hydrophobic fabrics (cloths)of plastics, e.g. polypropylene and polyester, are moderately pricedbecause of their large commercial demand as textiles and filters. Thesecloths offer the following advantages over the previous adsorptionsupports: they can accommodate a larger volume of sample per area; havea larger surface area for binding immunoreactants and forimmunoreactions; are easily washed because of minimum flow resistance;and have both strength and durability.

The enzyme immunoassay procedure for detecting antigens in test samplesusing antibody-coated macroporous hydrophobic cloths was found to befast and simple, requiring only the most basic instruments found in mostresearch and clinical laboratories. The enzyme immunoassay is alsodesigned so that a qualitative result can be obtained in field testsituations, where access to instrumentation is limited or non-existant.

The enzyme immunoassay procedure is a rapid and simple procedure whichcan be applied for the direct detection of antigens in test samples. Theassay can be easily and economically adapted for field testing, where apositive result could easily be distinguished from a negative one byvisual assessment of the substrate-indicator, (e.g. ABTS), whichproduces a blue-green colour in the presence of horseradish peroxidase.The "dipstick" format of the field kit makes the manipulation ofantibody-coated macroporous hydrophobic cloth throughout the proceduresimple and convenient, so that the test can be performed by untrainedhands. Furthermore, this format obviates the need for even commonlaboratory equipment, and all necessary reagents can be easily suppliedin the form of a kit.

Although horseradish peroxidase was used as an indicator, any othersuitable enzyme, e.g. alkaline phosphatase and β-galactosidase, can beemployed in the enzyme-antibody conjugate for the detection of antigens.Also, monovalent antigens e.g. toxins (i.e., haptens) can be detected bythe second embodiment of the enzyme immunoassay method. In this secondembodiment of the enzyme immunoassay, a standard preparation of themonovalent antigen could be immobilized on the macroporous hydrophobiccloth surface by adsorption or via a hydrophobic carrier. A test samplesuspected of harboring the antigen would then be mixed with anenzyme-antibody conjugate specific for that antigen and incubated withthe antigen-coated macroporous hydrophobic cloth. A negative control inwhich a representative sample devoid of antigen is mixed with theconjugate would be incubated with a separate antigen-coated macroporoushydrophobic cloth. Since the presence of free antigen in the test sampleshould prevent binding of the conjugate to the macroporous hydrophobiccloth surface, the enzyme immunoassay result would be obtained bycomparing the amount of enzyme immobilized on the test macroporous clothwith that obtained on the negative control macroporous hydrophobiccloth. Thus, the enzyme immunoassay is amenable to a variety of assayforms, the exact form being determined by the nature of the specificantigen being detected.

The results obtained indicate that several types of macroporoushydrophobic cloths can be used as solid phases for the adsorption ofantibodies. These include macroporous polypropylene macroporouspolyester, macroporous nylon, and macroporous polyethylene cloths, allof which were found to be suitable adsorbents for antibody, e.g.anti-Brucella antibody. All those macroporous hydrophobic cloths haveproven successful for the detection of antigens such as B. abortusantigens.

It has been found that whole bovine antiserum containing antibody withthe appropriate antigen-specificity can be used to coat macroporoushydrophobic cloth when heated at 75° C. for 10 minutes. This obviatesthe need for purified antibody preparations, which are time-consuming toproduce and may entail some expense. However, in order to minimize thepotential for cross-reactions it is preferred that enzyme-antibodyconjugates be prepared using purified antibodies. Since the conjugatecan be diluted up to 1,000 times, only a small amount of conjugate stockneed be prepared in this manner, thus maintaining the ease and economyof each individual test.

The detection of Brucella abortus (the causative agent of bovinebrucellosis) was used to test the utility of the method of thisinvention. Using the macroporous hydrophobic cloth carriers of thisinvention coated with whole antiserum preheated at 75° C. for 10minutes, the enzyme immunoassay was able to detect 0.3 nanograms of B.abortus lipopolysaccharide and 10⁴ B. abortus whole cells. Themacroporous polypropylene cloth-based enzyme immunoassay was alsosuccessfully adapted for the detection of bovine viral diarrhea (BVD)antigen.

As noted above Brucella abortus was used to examine the performance ofmacroporous hydrophobic cloth as adsorbents of immunoreactants. B.abortus causes brucellosis, a serious disease of humans and cattle.Confirmation of the brucellosis by the cultural diagnosis is a slow,complicated process of uncertain sensitivity. Rapid, simple andsensitive detection of Brucella antigens will facilitate confirmationand thus surveillance of brucellosis and its control.

Macroporous polypropylene cloth has been found to have excellentproperties as a solid phase in the enzyme immunoassay. The fact thatmacroporous polypropylene filter cloth is available in a nonwoven filtercloth form gives it the added advantage of retaining a stable fabricstructure (i.e., no loose edges) even under agitated conditions.Furthermore, macroporous polypropylene filter cloth is easily adaptedfor the preparation of commercial test kits.

The detection of B. abortus antigens by antibody-coated macroporoushydrophobic cloths is only one example of the method of this inventionfor the study of microbial antigen detection by cloth enzymeimmunoassay. The enzyme immunoassay method is amenable to the detectionof any given number of microbial antigens, provided that these aresufficiently small to be retained on the antibody-coated macroporoushydrophobic cloths throughout the enzyme immunoassay procedure. In caseswhere antigens, e.g. whole cells, are too large for effective retentionon the macroporous hydrophobic cloths, important antigenic componentsthereof might be dissociated from the surface by simple chemical ormechanical means so as to facilitate detection.

For example, the present invention is applicable to many immunologicallyreactive materials, e.g. proteins, peptides, polysaccharides, etc. whichare of decisive significance for an immunological determination, i.e.the presence of these materials is the determining factor in theimmunological test procedure. These materials can be detected in thebody fluids of humans and animals using immunological principles or canserve for their detection. Especially suitable immunologically-reactivematerials are pathogenic and vacultatively pathogenic organisms such as,for example, parasites, protozoa, bacteria or viruses or theirimmunologically active components, isolated antibodies from humans andanimals, serum constituents, toxins, hormones, enzymes, alkaloids, celland tissue extracts, substances with a small molecular weight such as,for example, insulin, anngiotensin and urokinase, biogenic amines, bloodcells, particles chemically or physically covered with antigens orantibodies, such as, for example, erythrocytes or latex particles.

The following Table provides a selection of typical diseases orconditions which can be determined with the aid of the immunoassaydevice in accordance with the present invention according to theimmunologically reactive materials lyophilised thereon.

                  TABLE                                                           ______________________________________                                        Antigen                Disease                                                ______________________________________                                        Toxoplasma gondii      Toxoplasmosis                                          Entamoeba histolytica  Amoebiasis                                             Trypanosoma cruzi      Chagas                                                 Trypanosoma gambiense/rhodesiense                                                                    Sleeping sickness                                      Leishmania donovani    Leishmaniasis                                          Schistosoma mansoni    Schistosomiasis                                        Echinococcus granulosus                                                                              Echinococcosis                                         Filariae               Filariasis                                             Fasciola hepatica      Fascioliasis                                           Plasmodia              Malaria                                                Candida species        Candidiasis                                            Aspergilli             Asperigillosis                                         Mycropolyspora faeni/Micromonospora                                                                  Farmer's lung                                          vulgaris                                                                      Treponema pallidum     Syphilis                                               Neisseria gonorrhoeae  Gonorrhea                                              Neissseria meningitis  Meningitis                                             Brucella abortus       Brucellosis                                            Mycoplasma pneumoniae  Pneumonia                                              Australia antigen      Acute hepatitis                                        Herpes simplex virus   Herpes simplex                                         Influenza virus        Flu                                                    Cell nuclei            Systemic lupus                                                                erythrematosis or                                                             Scleroderma                                            Cryptococci            Cryptococcosis                                         Torulopsis species     Systemic mycosis                                       H-antigen              Salmonella                                             (flagellar)                                                                   ______________________________________                                    

CONCLUSION

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Consequently, such changes and modifications are properly,equitably, and "intended" to be, within the full range of equivalence ofthe following claims.

What we claim is:
 1. An enzyme immunoassay device comprising thecombination of (a) a macroporous hydrophobic synthetic polymer woven ornon-woven cloth having a thickness of more than about 200 μm and havingspaces between fibres exceeding about 20 μm in diameter, the clothconsisting entirely of unmodified hydrophobic threads formed of asynthetic polymer selected from the group consisting of polypropylene,polyester, nylon, and polyethylene; and (b) an unmodified antibody or anunmodified antigen directly adsorbed thereon and directly absorbedtherein; the cloth having a Frazier Air Permeability in CFM/ft² at0.5"H₂ O of about 215 for a cloth of thickness about 40 mils, the cloththereby having such porosity that it can accommodate a large volume ofper surface area thereof, that it has a large surface area for bindingto the antibody or the antigen, respectively, and that it has minimumflow resistance.
 2. The enzyme immunoassay device of claim 1 whereinsaid antibody is provided by an antiserum containing an antibodyspecific for the antigen being tested.
 3. The enzyme immunoassay deviceof claim 2 wherein said antibody present in said antiserum have beenpartially denatured, by exposure to a low pH environment or by heating.4. The enzyme immunoassay device of claim 3 wherein said partialdenaturization is achieved.
 5. The enzyme immunoassay device of claim 4wherein said antibodies present in said antiserum have beenaffinity-purified.
 6. The enzyme immunoassay device of claim 1 whereinsaid antibody is provided by a purified antibody bearing the appropriatespecificity, which is either diluted or undiluted.
 7. The enzymeimmunoassay device of claim 1 in the form of said macroporoushydrophobic synthetic polymer cloth which is bonded to a differentmaterial, thereby to provide an antibody-coated test strip that may behandled throughout an enzyme assay procedure.
 8. An enzyme immunoassaymethod for detecting an antigen comprising the steps of: a) treating asurface of an immunoassay device comprising a macroporous hydrophobicsynthetic polymer woven or non-woven cloth having a thickness of morethan about 200 μm and having spaces between fibres exceeding about 20 μmin diameter, said cloth consisting entirely of unmodified hydrophobicthreads formed of a synthetic polymer selected from said groupconsisting of polypropylene, polyester, nylon, and polyethylene with anunmodified antibody, thereby to have an antibody directly adsorbedthereon and directly absorbed therein, said cloth having a Frazier AirPermeability in CFM/ft² at 0.5"H₂ O of about 215 for a cloth ofthickness about 40 mils, said cloth thereby having such porosity that itcan accommodate a large volume of liquid per surface area thereof, thatit has a large surface area for binding to said antibody and that it hasminimum flow resistance, thereby to provide an antibody surface-treatedcloth; b) applying, to said surface of said antibody surface-treatedcloth, an antigen being assayed, thereby to provide an antigen-treatedcloth; (c) incubating said antigen-treated cloth with a sample to betested for said antigen, thereby to provide an incubated cloth; d)washing said incubated cloth with a buffer to remove unadsorbedmaterial, thereby to provide a washed cloth; e) incubating said washedcloth with an enzyme-antibody conjugate prepared by coupling purifiedantibody specific for said antigen to a suitable indicator enzyme,thereby to provide an incubated washed cloth; f) washing said incubatedwashed cloth with a buffer to remove unreacted conjugate; and g)detecting enzyme-antibody conjugate remaining thereon by incubation in achromogenic substrate indicator solution to produce a visible colourupon product formation, indicative of said presence of an antigen
 9. Anenzyme immunoassay method for detecting an antibody comprising the stepsof: a) treating a surface of an immunoassay device comprising amacroporous hydrophobic synthetic polymer woven or non-woven clothhaving a thickness of more than about 200 μm and having spaces betweenfibres exceeding about 20 μm in diameter, said cloth consisting entirelyof unmodified hydrophobic threads formed of a synthetic polymer selectedfrom said group consisting of polypropylene, polyester, nylon, andpolyethylene with an unmodified antigen, thereby to have an antigendirectly adsorbed thereon and directly absorbed therein, said clothhaving a Frazier Air Permeability in CFM/ft² at 0.5"H₂ O of about 215for a cloth of thickness about 40 mils, said cloth thereby having suchporosity that it can accommodate a large volume of liquid per surfacearea thereof, that it has a large surface area for binding to saidantigen and that it has minimum flow resistance; thereby to provide anantigen surface-treated cloth; b) applying, to said surface of saidantigen surface-treated cloth, an antibody being assayed, thereby toprovide an antibody-treated cloth; c) incubating said antibody-treatedcloth with a sample to be tested for said antibody, thereby to providean incubated cloth; d) washing said incubated cloth with a buffer toremove unadsorbed material, thereby to provide a washed cloth; e)incubating said washed cloth with an enzyme-antibody conjugate preparedby coupling purified antibody specific for the test antibody to asuitable indicator enzyme, thereby to provide an incubated washed cloth;f) washing said incubated washed cloth with a buffer to remove unreactedconjugate; and g) detecting enzyme-antibody conjugate remaining thereonby incubation in a chromogenic substrate indicator solution to produce avisible colour upon product formation, indicative of said presence of anantibody.
 10. An enzyme immunoassay method for detecting an antibodycomprising the steps of: a) treating an immunoassay device comprising amacroporous hydrophobic synthetic polymer woven or non-woven clothhaving a thickness of more than about 200 μm and having spaced betweenfibres exceeding about 20 μm in diameter, said cloth consisting entirelyof unmodified hydrophobic threads formed of a synthetic polymer selectedfrom said group consisting of polypropylene, polyester, nylon, andpolyethylene with an unmodified antibody, thereby to have an antibodydirectly adsorbed thereon and directly absorbed therein, said clothhaving a Frazier Air Permeability in CFM/ft² at 0.5"H₂ O of about 215for a cloth of thickness about 40 mils, said cloth thereby having suchporosity that it can accommodate a large volume of liquid per surfacearea thereof, that it has a large surface area for binding to saidantibody and that it has minimum flow resistance, thereby to provide anantibody surface-treated cloth; b) applying to said surface of saidantibody surface-treated cloth, a mixture of said antibody being assayedand an enzyme-antigen conjugate specific for said antibody adsorbed ontosaid cloth, thereby to provide an antibody/enzyme-antigen conjugatetreated cloth; c) applying, to a surface of a control cloth identical tosaid hydrophobic synthetic polymer cloth, said same enzyme-antigenconjugate, thereby to provide an enzyme-antigen conjugate treatedcontrol cloth; d) incubating both said antibody/enzyme-antibodyconjugate treated cloth and said enzyme-antigen conjugate treatedcontrol cloth substantially simultaneously, to provide incubated cloths;e) washing both said incubated antibody/enzyme-antigen conjugate treatedcloth and said incubated enzyme-antigen conjugate treated control clothwith a buffer solution; and f) detecting said antibody by incubation ofboth said washed antibody/enzyme-antigen conjugate treated cloth andsaid washed enzyme-antigen conjugate treated control cloth in achromogenic substrate indicator solution to produce a visible colourupon product formation indicative of said presence of antibody, theamount of said antibody being determined by the difference in intensityof said colour on said antibody/enzyme-antigen conjugate treated clothand said colour on said enzyme-antigen conjugate treated control cloth.11. An enzyme immunoassay method for detecting an antigen comprising thesteps of: a) treating an immunoassay device comprising a macroporoushydrophobic synthetic polymer woven or non-woven cloth having athickness of more than about 200 μm and having spaced between fibresexceeding about 20 μm in diameter, said cloth consisting entirely ofunmodified hydrophobic threads formed of a synthetic polymer selectedfrom said group consisting of polypropylene, polyester, nylon, andpolyethylene with an unmodified antigen, thereby to have an antigendirectly adsorbed thereon and directly absorbed therein, said clothhaving a Frazier Air Permeability in CFM/ft² at 0.5"H₂ O of about 215for a cloth of thickness about 40 mils, said cloth thereby having suchporosity that it can accommodate a large volume of liquid per surfacearea thereof, that it has a large surface area for binding to saidantigen and that it has minimum flow resistance, thereby to provide anantigen surface-treated cloth; b) applying to said surface of saidantigen surface-treated cloth a mixture of said antigen being assayedand an enzyme-antibody conjugate specific for said antigen adsorbed ontosaid cloth, thereby to provide an antigen/enzyme-antibody conjugatetreated cloth; c) applying, to a surface of a control cloth identical tosaid hydrophobic synthetic polymer cloth, the same enzyme-antibodyconjugate, thereby to provide an enzyme-antibody conjugate treatedcontrol cloth; d) incubating both said antigen/enzyme-antibody conjugatetreated cloth and said enzyme-antibody conjugate treated control clothsubstantially simultaneously to provide incubated cloths; e) washingboth said incubated antigen/enzyme-antibody conjugate treated cloth andsaid incubated enzyme-antibody conjugate treated control cloth with abuffer solution; and f) detecting said antigen by incubation of bothsaid antigen/enzyme -antibody conjugate treated cloth and saidenzyme-antibody conjugate treated control cloth in a chromogenicsubstrate indicator solution to produce a visible colour upon productformation, indicative of the presence of said antigen, the amount ofsaid antigen being determined by the difference in intensity of thecolour on said antigen/enzyme-antibody conjugate treated cloth and thecolour on said enzyme-antibody conjugate treated control cloth.